Uncoupling complement C1s activation from C1q binding in apoptotic cell phagocytosis and immunosuppressive capacity

Abstract

Complement activation contributes to inflammation in many diseases, yet it also supports physiologic apoptotic cells (AC) clearance and its downstream immunosuppressive effects. The roles of individual complement components in AC phagocytosis have been difficult to dissect with artificially depleted sera. Using human in vitro systems and the novel antibody complement C1s inhibitor TNT003, we uncoupled the role of the enzymatic activation of the classical pathway from the opsonizing role of C1q in mediating a) the phagocytosis of early and late AC, and b) the immunosuppressive capacity of early AC. We found that C1s inhibition had a small impact on the physiologic clearance of early AC, leaving their immunosuppressive properties entirely unaffected, while mainly inhibiting the phagocytosis of late apoptotic/secondary necrotic cells. Our data suggest that C1s inhibition may represent a valuable therapeutic strategy to control classical pathway activation without causing significant AC accumulation in diseases without defects in AC phagocytosis.

Keywords: Apoptotic cells; C1q; C1s; Complement; Phagocytosis; Systemic lupus erythematosus.

Fig. 1. Blockade of the enzymatic C1s activity does not affect initial C1q binding to apoptotic cells

A) Early (A) (>65% Annexin V+PI−) and late (B) (>90% Annexin V+PI+) AC were incubated with serum-free media supplemented or not with purified C1q (15 μg/ml) and/or isotype control mAb (Iso C) or TNT003 (C1s inhibitor). C1q binding was monitored by flow cytometry and the results expressed as mean fluorescence intensity (MFI). Data was pooled from 3 independent experiments (n = 6–7 per condition for early AC; n = 4–5 per condition for late AC; p = NS difference with TNT003 or isotype control preincubation). C) Media containing NHS or NHS + C1q (15 μg/ml) were pre-incubated with isotype control mAb or TNT003 before incubation with late AC. The AC were then washed, and stained with a mouse-anti-human C1q mAb for flow cytometry analysis as in A. Data was pooled from 3 independent experiments (n = 4–5 wells per condition; p = NS change with TNT003 or isotype control pretreatment).

Fig. 2. C1s targeting prevents C3b deposition on late apoptotic cells

A) Media containing no serum, NHS, or HI NHS were incubated with early AC, followed by staining with mouse-anti human C3b mAb and flow cytometry analysis. The C3b MFI values were pooled from 4 independent experiments; n = 4–6 per condition; p = NS). B) Media containing no serum, NHS, or HI NHS were preincubated with either isotype control mAb (Iso C) or TNT003 before incubation with late AC, followed by C3b staining and flow cytometry analysis. The C3b MFI values were pooled from 8 independent experiments; n = 8–11 samples per condition; *p < 0.05).

Fig. 3. C1s blockade primarily inhibits the phagocytosis of late AC, while having a small effect on the uptake of early AC by human phagocytes

A) NHS or HI NHS containing either isotype control mAb (Iso C) or TNT003 were incubated with early AC. The AC were fed to M-CSF-derived human macrophages at a 2:1 apoptotic cell:macrophage ratio for 20 min at 37 °C. AC phagocytosis was quantified by calculating the percent inhibition of CFSE MFI of the CD14+ gated Mo population by isotype control mAb, TNT003, or C1qD serum with respect to the untreated wells. Data was pooled from 6 independent experiments with NHS or HI NHS (n = 12 per condition; **p < 0.01 between TNT003 and isotype control), and 3 experiments with C1qD serum (n = 6; *p < 0.05 between TNT003 and C1qD serum-mediated inhibition). B) Phagocytosis of late AC was assayed as in A. Data was pooled from 5 independent experiments (n = 6 wells per condition; ***p < 0.001 between TNT003 and isotype control).

Fig. 4. C1s blockade does not affect the immunosuppressive capacity of early apoptotic cells

Early AC pre-incubated with NHS and isotype control (Iso C) or TNT003 were added to human Mo (4:1 AC:Mo ratio) at the same time of LPS addition (1 ng/ml). After overnight culture, supernatants were harvested and [TNF-a] (A), [IL-6] (B) and [IL-1b] (C) were quantified by ELISA assay. Data is presented as percent [TNF-a], [IL-6], and [IL-1b] of LPS alone control well, and was pooled from 4 independent experiments (n = 4 per condition; **p < 0.01 for TNF and IL-6 inhibition by AC; ***p < 0.001 for IL-1b by two tailed paired Student’s t test. p = NS change in presence or absence of isotype control or TNT003 compared to untreated wells).